This invention relates to the use of magnetic or magnetizable particles, and, in particular, to methods and related devices used in mixing magnetic or (super)paramagnetic particles efficiently with a fluid and the separation of the magnetic particles from a fluid, optionally followed by resuspension of the particles in another fluid.
Magnetic particles are often used in separation processes. There are many biological assay methods and purification methods in which magnetic particles are used. For example, immuno assay methods, nucleic acid hybridization assays and the like. Magnetic particles can also be used in purification methods, to isolate particular compounds, proteins, nucleic acids, from the material in which they were contained. The particles can be used to separate certain components from a mixture, for example, because they are coated with a reagent with a specific affinity for the component. Magnetic particles can be drawn to, for example, the wall of a container in which the fluid with the magnetic particles was contained and the fluid can be removed and, optionally, be replaced with another fluid. Thus, the particles can be mixed with the fluid from which the specific component is to be removed, the component will bind to the magnetic particle, and a magnet can be used to separate the particles with the component from the remainder of the mixture in the fluid. Optionally the magnetic particles can be washed, and can be separated in another fluid. Or the component can be removed from the particles again into another fluid.
The use of magnetic particles for purifying a nucleic acid (NA) target from a biological sample is well known.
Purification methods for nucleic acid using magnetic particles have for example been described in EP757106 (Toyobo) and WO 96/41811 (Boehringer Mannheim). In these applications methods described wherein a sample solution containing nucleic acid is treated with a chaotropic substance to release the nucleic acid. After releasing the NA from the biological entity in the lysis buffer, the NA is bound to the magnetic particles. Both particles coated with a target-specific probe as well as particles having a metal oxide coating (e.g. silica), giving a generic binding of all NA contained in the sample are used for this purpose. After binding the target, interfering components such as cell debris, enzymes, proteins anti-coagulants and salt are removed by washing the magnetic particles in a (set of) wash buffer(s). Finally, the purified NA is released from the particles by mixing the particles in a small volume of elution buffer. This process is called elution since it is the nucleic acid that is eluted from the particles.
For efficient washing and elution the magnetic particles need to be well dispersed and mixed in the relevant buffers. In general, this washing and elution process may be hampered by the aggregation or clogging of the magnetic particles either caused by the adsorption on the magnetic particles of specific components in the lysed sample (e.g. g nomic DNA) or by residual magnetic dipole fields induced in the particles. In particular, the use of silica coated (magnetic) particles with samples that contain significant amounts of genomic DNA (whole blood, sputum, tissue), results in a tight pellet that is difficult to process.
Well-known methods for mixing (magnetic) beads in a liquid buffer are vortexing, sonification or pipetting. These methods however are difficult to automate, and/or give risk of sample to sample contamination by aerosol generation or they may degrade the NA target. Furthermore, these methods are not well suited for very small volumes of liquid (typically 0.01 ml) as may be required for the elution process.
A method and apparatus for separating and resuspending superparamagnetic particles is disclosed in WO 91/09308 (Diatec instruments).
In this application it was disclosed that superparamagnetic particles may be aggregated and resuspended by subsequent application of different magnetic fields. First and second applications of the magnetic field could be provided with the same magnet, which was then rotated around the container containing the particles to a different location. Two spaced opposed electromagnets, however, could also be used. These electromagnets were energized alternately to produce the first and second magnetic fields that keep the particles in suspension and mix them with the fluid in which they were contained. A method for the separation of magnetic particles from a fluid is disclosed in U.S. Pat. No. 3,985,649. The particles may be separated from a fluid by bringing the particles into close proximity with a magnet and moved trough the liquid along the wall of a container. They may even be moved out of the liquid in this way and can be transported to a second container. In U.S. Pat. No. 4,988,618 a device is described for use with assays wherein multiple small volume samples are tested at the same time. These type of assay can be performed in, for example, microtiter plates. Magnetic microparticles are present in each well of the microtiter plate. The device thus has multiple orifices and the orifices are each surrounded by multiple permanent magnets, preferably four. The resulting structure of magnets and orifices is rigid; the magnets are not intended to be moved and are mounted in fixed relations with respect to themselves and to the base of the device. All magnets are aligned and the field orientation of the magnets may be such that all magnets have the same field direction or neighboring magnets have opposite field directions. The magnet orientation thus results in four spot attraction sites per orifice. The magnets are purely meant for separation purposes. It is disclosed in the patent that the device may further comprise means for agitating the reagents within the containers.
The present invention relates to a method and device, which allows efficient mixing of magnetic or magnetizable particles in a fluid, and optionally separation of the particles from said fluid. Use is made of magnetic field of opposite and changing directions. It has been found that, when magnetic or magnetizable particles in a fluid are subjected to these magnetic fields, the particles are, under the influence of the field, efficiently contacted with the fluid. Such particles normally may tend to form a clot, which can prevent efficient mixing with a fluid. It has been found that, by subjecting the container in which the fluid and the particles are comprised, to magnetic fields of different and changing directions, the particles are efficiently separated from each other and drawn trough the fluid in such a way that a very efficient mixing process occurs. The method allows efficient mixing of particles with even very small fluid volumes. The method of the invention therefore has the advantage that it may save in, for example, washing fluids and may allow the reduction of the volume of fluid needed. Thus, for example is isolation procedures, the method of the invention allows the purification of reagents in high concentrations. Beside, whereas prior art methods can be laborious and time consuming, the method is fast and easy to perform.
The method and device according to the invention are especially suitable for use with isolation procedures, where, usually an ingredient is to be isolated in rather pure form from a relatively large volume of sample fluid, and concentrated into a smaller volume of another fluid to be suitable for further use. In the case of a method for the isolation of nucleic acid such further use may be a nucleic acid amplification method or an assay for the detection of nucleic acid or both.
Thus, provided with the invention is a method of mixing, in one or more container(s), magnetic or (super)paramagnetic particles with a fluid, using more than one magnets, whereby the containers are subjected to magnetic fields with different and changing directions by moving the magnets with respect to the position of the container(s) and/or by moving the containers with respect to the positions of the magnets.
With xe2x80x9cmixingxe2x80x9d in this context is meant that the particles and the fluid are brought in close contact. Mixing thus, means xe2x80x9ccontactingxe2x80x9d in a very efficient manner, such as when particles would be washed or reacted with components present in the fluid. Mixing, in this context, does not necessarity provide a homogeneous mixture after the process is finished. The particles may, when the magnets are removed, segregate to the bottom of the container in which they are comprised or may be held to the wall of the container in a particular location by the magnets. The mixing process can for example be used to wash the particles or to react the particles with a component of the liquid, or to bind a component of the liquid to a reagent coated on the particles. Likewise, the mixing process may result in the elution of a certain component originally present on the particles into the surrounding liquid. The method of the invention is applicable in each of these processes and provides an efficient rapid and convenient way of contacting magnetic or magnetizable particles with a volume of a certain fluid.
The present invention thus provides a generic method for mixing magnetic particles with a fluid almost independent of their level of pelleting/aggregation. The method further allows releasing of reagents bound to the particles, for example nucleic acid, from the particles and concentration into a small volume. The method is easy to automate and well suited for high throughput formats. It minimizes the risk of contamination by droplets or aerosols.